The present invention is a method of determining the sequences of natural killer cell immunoglobulin-like receptor or “KIR” genes within a single individual or within each one of simultaneously tested multiple individuals.
Natural Killer Cells
Natural Killer (NK) cells are part of the innate immune system and are specialized for early defense against infection as well as tumors. The NK cells were first discovered as a result of their ability to kill tumor cell targets. Unlike cytolytic T-cells, NK cells can kill targets in a non-major histocompatibility complex (non-MHC)-restricted manner. As an important part of the innate immune system, the NK cells comprise about 10% of the total circulating lymphocytes in the human body.
Because of their ability to kill other cells, NK cells are normally kept under tight control. All normal cells in the body express the MHC class I molecules on their surface. These molecules protect normal cells from killing by the NK cells because they serve as ligands for many of the receptors found on NK cells. Cells lacking sufficient MHC class I on their surface are recognized as ‘abnormal’ by NK cells and killed. Simultaneously with killing the abnormal cells, the NK cells also elicit a cytokine response.
Natural killer cells constitute a rapid-response force against cancer and viral infections. These specialized white blood cells originate in the bone marrow, circulate in the blood, and concentrate in the spleen and other lymphoid tissues. NK cells key their activities on a subset of the human leukocyte antigen (HLA) proteins that occur on the surfaces of healthy cells but that virus- and cancer-weakened cells shed. The HLA proteins are encoded by Major Histocompatibility Complex (MHC) genes. When NK cells encounter cells that lack HLA proteins, they attack and destroy them—thus preventing the cells from further spreading the virus or cancer. NK cells are distinguished from other immune system cells by the promptness and breadth of their protective response. Other white blood cells come into play more slowly and target specific pathogens—cancers, viruses, or bacteria—rather than damaged cells in general.
KIR Genes
The natural killer cell immunoglobulin-like receptor (KIR) gene family is one of several families of receptors that encode important proteins found on the surface of natural killer (NK) cells. A subset of the KIR genes, namely the inhibitory KIR, interact with the HLA class I molecules, which are encoded within the human MHC. Such interactions allow communication between the NK cells and other cells of the body, including normal, virally infected, or cancerous cells. This communication between KIR molecules on the NK cells and HLA class I molecules on all other cells, helps determine whether or not cells in the body are recognized by the NK cells as self or non-self. Cells which are deemed to be ‘non-self’ are targeted for killing by the NK cells.
KIR Gene and Protein Structure
The KIR gene family consists of 16 genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.) The KIR gene cluster is located within a 100-200 kb region of the Leukocyte Receptor Complex (LRC) located on chromosome 19 (19q13.4) The gene complex is thought to have arisen by gene duplication events occurring after the evolutionary split between mammals and rodents The KIR genes are arranged in a head-to tail fashion, with only 2.4 kb of sequence separating the genes, except for one 14 kb sequence between 3DP1 and 2DL4. Because the KIR genes arose by gene duplication, they are very similar in sequence, showing 90-95% identity with one another. Human individuals differ in the number and type of KIR genes that they inherit; the KIR genotype of individuals and within ethnic groups can be quite different. At the chromosomal level, there are two distinct types of KIR haplotypes (See FIG. 1, adapted from Martin et al. Immunogenetics. (2008) December; 60(12):767-774). The A-haplotype contains no stimulatory genes (2DS and 3DS1) other than 2DS4, no 2DL5 genes and no 2DL2 genes. The B-haplotype is more variable in gene content, with different B-haplotypes containing different numbers of stimulatory genes, either one or two 2DL5 genes, etc. (Martin M P, et al. (2008) KIR haplotypes defined by segregation analysis in 59 Centre d'Etude Polymorphisme Humain (CEPH) families. Immunogenetics, December; 60(12):767-774.).
All of the KIR proteins are anchored to the cell membrane, with either two or three extracellular immunoglobulin-like domains and a cytoplasmic tail. Nine KIR genes (KIR2DL and KIR3DL) encode proteins with long cytoplasmic tails that contain immune tyrosine-based inhibitory motifs (ITIM). These KIR proteins can send inhibitory signals to the natural killer cell when the extra-cellular domain has come into contact with its ligand. The remaining KIR genes encode proteins with short cytoplasmic tails. These proteins send activating signals via adaptor molecules like DAP 12.
KIR receptor structure and the identity of the HLA class I ligands for each KIR receptor are shown on FIG. 2 (adapted from Parham P. et al., Alloreactive killer cells: hindrance and help for hematopoietic transplants. Nature Rev. Immunology. (2003)3:108-122.) The nomenclature for the killer-cell immunoglobulin-like receptors (KIRs) describes the number of extracellular immunoglobulin-like domains (2D or 3D) and the length of the cytoplasmic tail (L for long, S for short). Each immunoglobulin-like domain is depicted as a loop, each immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail as an oblong shape, and each positively charged residue in the transmembrane region as a diamond. The stimulatory KIR are noted in italics. (Parham P. et al., (2003).
The strength of the interactions between the KIR and their HLA class I ligands can be dependent upon both the KIR sequence and the HLA sequence. While the HLA region has been studied for over 40 years, the KIR molecules were first described (as NKB1) in the mid-1990s (Lanier et al. (1995) The NKB1 and HP-3E4 NK cells receptors are structurally distinct glycoproteins and independently recognize polymorphic HLA-B and HLA-C molecules. J Immunol. April 1; 154(7):3320-3327 and Litwin V. et al. (1994) NKB1: a natural killer cell receptor involved in the recognition of polymorphic HLA-B molecules. J Exp Med. August 1; 180(2):537-543). The first years of discovery were mainly devoted to describing the different KIR genes, and methods were developed to determine individual KIR genotypes. Utilizing these methods, KIR gene associations with autoimmune disease and recipient survival after allogeneic hematopoietic cell transplantation have been shown (Parham P. (2005) MHC class I molecules and KIRs in human history, health and survival. Nature reviews, March; 5(3):201-214). It is now clear that each KIR gene has more than one sequence; that is, each KIR gene has variable sequence because of single nucleotide polymorphisms (SNPs), and in some instances, insertions or deletions within the coding sequence. Studies have shown that KIR3DL1 polymorphism can affect not only the expression levels of KIR3DL1 on natural killer cells, but also the binding affinity of KIR3DL1 to its ligand.
KIR Association with Disease
Studies designed to investigate the role of KIR in human disease have shown an association with various KIR genes and viral infections such as CMV, HCV and HIV, autoimmune diseases, cancer and preeclampsia (Parham P. (2005) MHC class I molecules and KIRs in human history, health and survival. Nature reviews, March; 5(3):201-214). In a recent study on genetic susceptibility to Crohn's disease, an inflammatory autoimmune bowel disease, it was found that patients who are heterozygous for KIR2DL2 and KIR2DL3 and homozygous for the C2 ligand are susceptible to disease, whereas the C1 ligand is protective. (Hollenbach J A et al. (2009) Susceptibility to Crohn's Disease is mediated by KIR2DL2/KIR2DL3 heterozygosity and the HLAC ligand. Immunogenetics. October; 61(10):663-71). Other studies on KIR and unrelated hematopoietic cell transplantation (HCT) for Acute Myeloid Leukemia (AML) have shown a significantly higher 3 year overall survival rate and a 30% overall improvement in the risk of relapse-free survival with B/x donors compared to A/A donors. (Cooley S, et al. (2009) Donors with group B KIR haplotypes improve relapse-free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia. Blood. January 15; 113(3):726-732; and Miller J S, et al. (2007) Missing KIR-ligands is associated with less relapse and increased graft versus host disease (GVHD) following unrelated donor allogeneic HCT. Blood, 109(11):5058-5061). Such studies have been performed with knowledge of the KIR genotype of patients and controls, but have not been performed for KIR at an allelic level. Much as specific HLA alleles have been shown to be important in human disease, (for example HLA-DR3 and HLA-DR4 association with type I diabetes and HLA-DR8 with juvenile rheumatoid arthritis) the ability to genotype KIR at the allelic level will refine studies associating KIR with human disease.